Expression Of Pectinase Genes Research Articles

VmMon1-Ccz1 Complex Is Required for Conidiation, Autophagy, and Virulence in Valsa mali.

Apple Valsa canker caused by Valsa mali is a serious disease in eastern Asia, especially in China. In our previous proteomics study, monensin sensitivity 1 protein in Valsa mali (VmMon1) was identified to be significantly upregulated during V. mali infection. It was reported Mon1 protein formed a heterodimer called MC (Mon1-Ccz1) complex with caffeine, calcium, and zinc sensitivity 1 protein (Ccz1) in yeast. However, Ccz1 had not been identified in plant-pathogenic fungi such as Fusarium graminearum and Magnaporthe oryzae. Here, we identified a Ccz1 ortholog VmCcz1 in V. mali, by using DELTA-BLAST. The interaction of VmMon1 and VmCcz1 were verified using yeast two-hybrid assay, bimolecular fluorescence complementation, and co-immunoprecipitation assays. Further yeast three-hybrid screenings determined that VmRab7 (Ras-related protein in V. mali) interacted with the MC complex. Targeted gene deletion showed that the ∆VmMon1 and ∆VmCcz1 mutants were defective in vegetative growth, conidiation, and pathogenicity. In addition, both mutants were more sensitive to osmotic and oxidative stresses and intracellular protein transport inhibitors. Cytological examination revealed that the ∆VmMon1 and ∆VmCcz1 mutants were impaired in vacuole fusion and autophagy. More importantly, expression of pectinase genes decreased in both mutants compared with those of the wild type during infection. Overall, our study identified Mon1 and Ccz1 genes in V. mali and provided evidence that VmMon1 and VmCcz1 are critical components that modulate vacuole fusion and autophagy, thereby affecting the development, conidiation, and pathogenicity of V. mali. [Formula: see text] Copyright © 2022 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Understanding the role of the master regulator, PecS, in Agrobacterium fabrum

Bacterial plant pathogens have devised means of surviving in a hostile environment created by their hosts during the pathogenic phase of their life cycle. Critical to this survival mechanism are a family of transcriptional regulators known as Multiple antibiotic resistance Regulators (MarR), known to regulate the expression of genes involved in virulence, stress response, multidrug resistance etc. Recent studies have associated PecS, a member of the MarR family, as a master regulator of virulence gene expressions in the Enterobacterium Dickeya dadantii, a soft rot-causing phytopathogen of a range of plant species. D. dadantii PecS controls both the expression of pectinase genes and biosynthesis of a blue pigment known as indigoidine (an antioxidant). PecS has also been characterized in other plant pathogens such as, Pectobacterium atrosepticum, and it has been identified in the human pathogens, Klebsiella pneumoniae and Vibrio vulnificus. However, the role ofPecS in the crown gall-causing Agrobacterium fabrum remains largely unknown. To understand the role of A. fabrum PecS in the physiology of the bacterium, plant association, and promotion of survival in response to host defenses, we created a pecS knockout and determined growth, motility, biofilm and stress response. Our result shows a higher growth rate in pecS knockoutthan the Wild Type over a 14 hour period, while the Wild Type was more motile after 48 hours of incubation in a 0.3% (wt/vol) agar. Biofilm formation was measured at 24, 48 and 72 hours, using crystal violet staining, and the data shows that Wild Type forms more biofilm than the knockout at all the time points. Stress response assays show the Wild Type was more sensitive to 5 mM of both cumene hydroperoxide and hydrogen peroxide, 10 mM tert-butyl hydroperoxide and 25 ug ampicillin compared to the knockout strain, while the knockout was more sensitive to 25 ug and 200 ug trimethoprim and streptomycin respectively. The study expands our knowledge about the master regulatory role of A. fabrum PecS and proves that A. fabrum PecSis implicated in bacterial physiology, plant association and survival.

Baseline sensitivity and action mechanism of the sterol demethylation inhibitor flusilazole to Valsa mali

The apple Valsa canker caused by Valsa mali is a devastating branch disease that has seriously threatened the development of the apple industry worldwide. In current study, a total of 115 V. mali strains collected from different apple orchards in Shaanxi Province of China during 2016 and 2017 were tested for their sensitivity to flusilazole. The average EC50 (effective concentrations causing 50% mycelial growth inhibition) value of all tested strains for flusilazole was 0.0892 (±0.0036) μg/mL and the frequency distribution of the EC50 values was unimodal. Flusilazole exhibited both excellent protective and curative activity on detached apple branches, which was significantly better than the commonly used fungicide thiophanate-methyl. After flusilazole treatment, mycelia twisted with offshoot of top increased, the V. mali strains lost the ability of fruiting body production, and cell membrane permeability of the mycelia increased while ergosterol content and pectinase activity decreased. The expression of pectinase genes involved in virulence down-regulated after flusilazole treatment. This study is the first report on the baseline sensitivity of V. mali to flusilazole. These results indicated that flusilazole has a great potential to play an important role in the management of Valsa canker.

Adaptive regulation of virulence genes by microRNA-like RNAs in Valsa mali.

MicroRNAs play important roles in the regulation of gene expression in plants and animals. However, little information is known about the action mechanism and function of fungal microRNA-like RNAs (milRNAs). In this study, combining deep sequencing, molecular and histological assays, milRNAs and their targets in the phytopathogenic fungus Valsa mali were isolated and identified. A critical milRNA, Vm-milR16, was identified to adaptively regulate the expression of virulence genes. Fourteen isolated milRNAs showed high expression abundance. Based on the assessment of a pathogenicity function of these milRNAs, Vm-milR16 was found to be a critical milRNA in V.mali by regulating sucrose non-fermenting 1 (VmSNF1), 4,5-DOPA dioxygenase extradiol (VmDODA), and a hypothetical protein (VmHy1). During V.mali infection, Vm-milR16 is downregulated, while its targets are upregulated. Overexpression of Vm-milR16, but not mutated Vm-milR16, significantly reduces the expression of targets and virulence of V.mali. Furthermore, deletion of VmSNF1, VmDODA and VmHy1 significantly reduce virulence of V.mali. All three targets seem to be essential for oxidative stress response and VmSNF1 is required for expression of pectinase genes during V.mali-host interaction. Our results demonstrate Vm-milRNAs contributing to the infection of V.mali on apple trees by adaptively regulating virulence genes.

VmPacC Is Required for Acidification and Virulence in Valsa mali.

The role of the transcription factor PacC has been characterised in several pathogenic fungi, and it affects virulence via several mechanisms. In this study, we examined the role of the PacC homolog VmPacC in Valsa mali, the causal agent of apple canker disease. We found that the expression of VmPacC was up-regulated in neutral and alkaline pH and during infection. At pH 6–10, the radial growth of a VmPacC deletion mutant decreased compared to wild-type. In addition, the sensitivity to oxidative stress of the VmPacC deletion mutant was impaired, as its growth was more severely inhibited by H2O2 than that of the wild-type. The lesion size caused by the VmPacC deletion mutant was smaller than that of the wild-type on apple leaves and twigs. Interestingly, expression of pectinase genes increased in deletion mutant during infection. To further confirm the negative regulation, we generated dominant activated C-27 allele mutants that constitutively express VmPacC. The pectinase activity of activated mutants was reduced at pH 4. We further observed that V. mali can acidify the pH during infection, and that the capacity for acidification was impaired after VmPacC deletion. Furthermore, VmPacC is involved in the generation of citric acid, which affects virulence. These results indicate that VmPacC is part of the fungal responses to neutral and alkaline pH and oxidative stress. More importantly, VmPacC is required for acidification of its environment and for full virulence in V. mali.

Transcription factor VmSeb1 is required for the growth, development, and virulence in Valsa mali

Transcription factor Seb1 contains two C2H2 zinc finger motifs which are similar to the Msn2/4 of Saccharomyces cerevisiae. The homologous proteins of Seb1 function to regulate the response to various stresses or decomposing and utilizing pectin in some fungi. In this study, we characterized a homologue of Seb1 gene, VmSeb1, in Valsa mali, which causes a highly destructive bark disease on apple. VmSeb1 deletion mutant showed a drastic reduction in growth rate in vitro. It is also important for conidiation because VmSeb1 deletion mutant formed more pycnidia on PDA medium. Deletion mutant of VmSeb1 increased melanin genes expression. In addition, the sensitivity to oxidative stress increased and cell wall inhibitor in VmSeb1 deletion mutant, as its growth was more severely inhibited by H2O2 and Congo red than that in the wild-type. The virulence assay showed that the lesion length caused by the VmSeb1 deletion mutant was smaller compared to wild-type on detached apple twigs. However, expression of pectinase genes and pectinase activity in deletion mutant were the same as those of the wild-type during infection. These results indicate that VmSeb1 plays important roles in growth, asexual development, response to oxidative stress, maintenance of cell wall integrity, and virulence. However, VmSeb1 is not involved in the regulation of pectinase genes expression in V. mali.

The cyclic AMP receptor protein is the main activator of pectinolysis genes in Erwinia chrysanthemi.

The main virulence factors of the phytopathogenic bacterium Erwinia chrysanthemi are pectinases that cleave pectin, a major constituent of the plant cell wall. Although physiological studies suggested that pectinase production in Erwinia species is subjected to catabolite repression, the direct implication of the cyclic AMP receptor protein (CRP) in this regulation has never been demonstrated. To investigate the role of CRP in pectin catabolism, we cloned the E. chrysanthemi crp gene by complementation of an Escherichia coli crp mutation and then constructed E. chrysanthemi crp mutants by reverse genetics. The carbohydrate fermentation phenotype of the E. chrysanthemi crp mutants is similar to that of an E. coli crp mutant. Furthermore, these mutants are unable to grow on pectin or polygalacturonate as the sole carbon source. Analysis of the nucleotide sequence of the E. chrysanthemi crp gene revealed the presence of a 630-bp open reading frame (ORF) that codes for a protein highly similar to the CRP of E. coli. Using a crp::uidA transcriptional fusion, we demonstrated that the E. chrysanthemi CRP represses its own expression, probably via a mechanism similar to that described for the E. coli crp gene. Moreover, in the E. chrysanthemi crp mutants, expression of pectinase genes (pemA, pelB, pelC, pelD, and pelE) and of genes of the intracellular part of the pectin degradation pathway (ogl, kduI, and kdgT), which are important for inducer formation and transport, is dramatically reduced in induced conditions. In contrast, expression of pelA, which encodes a pectate lyase important for E. chrysanthemi pathogenicity, seems to be negatively regulated by CRP. The E. chrysanthemi crp mutants have greatly decreased maceration capacity in potato tubers, chicory leaves, and celery petioles as well as highly diminished virulence on saintpaulia plants. These findings demonstrate that CRP plays a crucial role in expression of the pectinolysis genes and in the pathogenicity of E. chrysanthemi.

ChemInform Abstract: Synthesis of New Methyl Esters of 3‐Deoxy‐D‐erythro‐2‐hexulosonic Acid (KDG) Analogs: Inducers of the Expression of Pectinase Genes in Bacteria Erwinia Chrysanthemi

AbstractChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 100 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a “Full Text” option. The original article is trackable via the “References” option.

ChemInform Abstract: Synthesis of Methyl Esters of 3‐Deoxy‐D‐erythro‐2‐hexulosonic Acid ( KDG) Analogues (IV) and (VI), Inducers of the Expression of Pectinase Genes in Bacteria Erwinia chrysanthemi.

AbstractChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 100 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a “Full Text” option. The original article is trackable via the “References” option.

Synthesis of methyl esters of 3-deoxy-d-erythro-2-hexulosonic acid (KDG) analogs, inducers of the expression of pectinase genes in bacteria Erwinia chrysanthemi

Two title compounds, methyl 3-deoxy-l-threo-2-hexulosonate 3 and methyl 3,5-dideoxy-d-glycero-2-hexulosonate 4, were prepared from d-glucono-1,5-lactone and proved to be gratuitous inducers of the expression of pectinase genes in the phytopathogenic bacteria Erwinia chrysanthemi.

Inducing properties of analogs of 2-keto-3-deoxygluconate on the expression of pectinase genes of Erwinia chrysanthemi

In Erwinia chrysanthemi, all the genes involved in pectin degradation are controlled by the negative regulatory gene kdgR. 2-keto-3-deoxy-gluconate (KDG) is the inducing molecule that interacts with KdgR to allow the expression of all the genes of the kdg regulon. The inducing properties on the expression of genes regulated by kdgR of various analogs and derivatives of KDG were tested. All the inducers share the common moiety COOH-CO-CH2-CHOH-C-C included in a pyranic cycle. Our results show that esterification of C1 prevents induction. Presence of a ketone function on C2 and absence of hydroxyl on C3 are necessary for induction. The nature and the configuration of substituent on C5 has no influence on induction. Two compounds have interesting properties: 5-O-methyl-KDG is a gratuitous inducer, and gluconic acid can prevent induction.

Inducing properties of analogs of 2-keto-3-deoxygluconate on the expression of pectinase genes ofErwinia chrysanthemi

In Erwinia chrysanthemi, all the genes involved in pectin degradation are controlled by the negative regulatory gene kdgR. 2-keto-3-deoxy-gluconate (KDG) is the inducing molecule that interacts with KdgR to allow the expression of all the genes of the kdg regulon. The inducing properties on the expression of genes regulated by kdgR of various analogs and derivatives of KDG were tested. All the inducers share the common moiety COOH-CO-CH2-CHOH-C-C included in a pyranic cycle. Our results show that esterification of C1 prevents induction. Presence of a ketone function on C2 and absence of hydroxyl on C3 are necessary for induction. The nature and the configuration of substituent on C5 has no influence on induction. Two compounds have interesting properties: 5-O-methyl-KDG is a gratuitous inducer, and gluconic acid can prevent induction.